The human MAP Kinase-interacting kinases, also known as MAP Kinase signal-integrating kinases, (MNKs), are ubiquitously expressed protein-serine/threonine kinases that are directly activated by ERK or p38 MAP kinases (Buxade, M.; Parra-Palau, J. L.; Proud, C. G. Front Biosci. 2008, 13, 5359-5373; Fukunaga, R.; Hunter, T. EMBO J. 1997, 16, 1921-1933; Waskiewicz, A. J.; Flynn, A.; Proud, C. G.; Cooper, J. A. EMBO J. 1997, 16, 1909-1920). They comprise a group of four proteins derived from two genes (gene symbols: MKNK1 and MKNK2) by alternative splicing. MNK1a/b and MNK2a/b proteins differ at their C-termini, in each case the a-form has a longer C-terminal region than the b-form which lacks the MAP Kinase-binding region. The N-termini of all forms contains a polybasic region which binds importin α and the translation factor scaffold protein eukaryotic Initiation Factor (eIF4G). The catalytic domains of MNK1a/b and MNK2a/b share three unusual features, two short inserts and a DFD motif instead of the most common DFG tripeptide present found on other kinases. MNK isoforms differ markedly in their activity, regulation and subcellular localization. The best-characterized MNK substrate is Eukaryotic Initiation Factor-4 E (eIF4E). Although the cellular role of eIF4E phosphorylation remains unclear and is thought to promote export of a defined set of mRNAs from the nucleus. Other MNK substrates bind to AU-rich elements that modulate the stability/translation of specific mRNAs. MNK1 is highly expressed in hematological malignancies, and both MNK1 and MNK2 are up-regulated in solid tumors such as gliomas and ovarian cancers (Worch, J.; Tickenbrock, L.; Schwable, J.; Steffen, B.; Cauvet, T.; Mlody, B.; Buerger, H.; Koeffler, H. P.; Berdel, W. E.; Serve, H.; Muller-Tidow, C. Oncogene 2004, 23, 9162-9172; Pellagatti, A.; Esoof, N.; Watkins, F.; Langford, C. F.; Vetrie, D.; Campbell, L. J.; Fidler, C.; Cavenagh, J. D.; Eagleton, H.; Gordon, P.; Woodcock, B.; Pushkaran, B.; Kwan, M.; Wainscoat, J. S.; Boultwood, J. Br. J. Haematol. 2004, 125, 576-583; Bredel, M.; Bredel, C.; Juric, D.; Harsh, G. R.; Vogel, H.; Recht, L. D.; Sikic, B. I. Cancer Res. 2005, 65, 4088-4096; Hendrix, N. D.; Wu, R.; Kuick, R.; Schwartz, D. R.; Fearon, E. R.; Cho, K. R. Cancer Res. 2006, 66, 1354-1362).
eIF4E regulates the expression of genes involved in the proliferation and survival as a cap dependent mRNA translation and mRNA export factor. eIF4E is dysregulated in several human cancers, including breast, prostate, and leukemia, and elevated levels of eIF4E are a marker of poor prognosis (Nathan, C. O.; Carter, P.; Liu, L.; Li, B. D.; Abreo, F.; Tudor, A.; Zimmer, S. G.; De Benedetti, A. Oncogene 1997, 15, 1087-1094; Bianchini, A.; Loiarro, M.; Bielli, P.; Busa, R.; Paronetto, M. P.; Loreni, F.; Geremia, R.; Sette, C. Carcinogenesis 2008, 29, 2279-2288.; Topisirovic, I.; Guzman, M. L.; McConnell, M. J.; Licht, J. D.; Culjkovic, B.; Neering, S. J.; Jordan, C. T.; Borden, K. L. Mol. Cell Biol. 2003, 23, 8992-9002; Graff, J. R.; Zimmer, S. G. Clin. Exp. Metastasis 2003, 20, 265-273). Moreover, overexpression and dysregulation of eIF4E leads to an increased number of tumors, invasion, and metastases in mouse models13 and transgenic expression of eIF4E leads to a variety of cancers (Graff, J. R.; Zimmer, S. G. Clin. Exp. Metastasis 2003, 20, 265-273; Ruggero, D.; Montanaro, L.; Ma, L.; Xu, W.; Londei, P.; Cordon-Cardo, C.; Pandolfi, P. P. Nat. Med. 2004, 10, 484-486). eIF4E overexpression is believed to increase the translation of weakly competitive mRNAs, many of which encode products that stimulate cell growth and angiogenesis, e.g., fibroblast growth factor 2 and vascular endothelial growth factor, cyclin D1, and ribonucleotide reductase (Kevil, C.; Carter, P.; Hu, B.; DeBenedetti, A. Oncogene 1995, 11, 2339-2348; Kevil, C. G.; De Benedetti, A.; Payne, D. K.; Coe, L. L.; Laroux, F. S.; Alexander, J. S. Int. J. Cancer 1996, 65, 785-790; Scott, P. A.; Smith, K.; Poulsom, R.; De Benedetti, A.; Bicknell, R.; Harris, A. L. Br. J. Cancer 1998, 77, 2120-2128; Rosenwald, I. B.; Lazaris-Karatzas, A.; Sonenberg, N.; Schmidt, E. V. Mol. Cell Biol. 1993, 13, 7358-7363; Abid, M. R.; Li, Y.; Anthony, C.; De Benedetti, A. J. Biol. Chem. 1999, 274, 35991-35998). eIF4E is phosphorylated by the MNK1/2 serine/threonine kinases in response to activation by mitogenic and stress signals downstream of ERK1/2 and p38 MAP kinase respectively (Buxade, M.; Parra-Palau, J. L.; Proud, C. G. Front Biosci. 2008, 13, 5359-5373; Fukunaga, R.; Hunter, T. EMBO J. 1997, 16, 1921-1933; Waskiewicz, A. J.; Flynn, A.; Proud, C. G.; Cooper, J. A. EMBO J. 1997, 16, 1909-1920). Thus, inhibitors of MNK1/2 will prevent eIF4E phosphorylation and therefore could provide a viable therapeutic approach in high-eIF4E dependent cancers.
Studies have shown that overexpression of eIF4E, as well as eIF4E phosphorylation, promote cancer cell survival, at least in part by increasing the level of the anti-apoptotic protein Mel-1 (Wendel, H. G.; Silva, R. L.; Malina, A.; Mills, J. R.; Zhu, H.; Ueda, T.; Watanabe-Fukunaga, R.; Fukunaga, R.; Teruya-Feldstein, J.; Pelletier, J.; Lowe, S. W. Genes Dev. 2007, 21, 3232-3237; Ueda, T.; Watanabe-Fukunaga, R.; Fukuyama, H.; Nagata, S.; Fukunaga, R. Mol. Cell Biol. 2004, 24, 6539-6549). Mcl-1 is a Bcl2 family member with a very short half-life, and Mel-1 mRNA translation highly depends on eIF4E. Thus, it is foreseeable that the inhibition of eIF4E phosphorylation by MNK might induce tumor cells death, as shown for Myc-induced lymphoma. (Wendel, H. G.; Silva, R. L.; Malina, A.; Mills, J. R.; Zhu, H.; Ueda, T.; Watanabe-Fukunaga, R.; Fukunaga, R.; Teruya-Feldstein, J.; Pelletier, J.; Lowe, S. W. Genes Dev. 2007, 21, 3232-3237) Blast crisis chronic myeloid leukemia (BC-CML) is characterized by an expansion of a population of granulocyte macrophage progenitor-like cells (GMPs) that have acquired self-renewal capacity, a feature not seen in normal or chronic phase (CP) GMPs and targeting of the MNK-eIF4E axis in blast crisis chronicmyeloid leukemia inhibits leukemia stem cell function. (Sharon Lim, Tzuen Yih Saw, Min Zhang, Matthew R. Janes, Kassoum Nacro, Jeffrey Hill, An Qi Lim, Chia-Tien Chang, David A. Fruman, David A. Rizzieri, Soo Yong Tan, Hung Fan, c, Charles T. H. Chuah, g, and S. Tiong Ong; N. Proc. Natl. Acad. Sci. U.S.A 2013, 110(25), E2298-E2307). The ability to self-renew is thought to be mediated by β-catenin activation, and may contribute to disease persistence, as well as initiate drug resistance. It was found siRNA-mediated knockdown or inhibition of the MNK1/2 kinases (which mediate in vivo eIF4E phosphorylation) with small molecules prevented the increased beta-catenin activity, induced by eIF4E overexpression. These studies suggest that pharmacologic inhibition of the MNK1/2 kinases is a plausible therapeutic mean to treat BC CML.
The level of expression of eIF4E and the degree of eIF4E phosphorylation is regulated by pathways that include the P38 kinase, MAPK kinase and AKT/mTOR pathways (Hay, N. Proc. Natl. Acad. Sci. U.S.A 2010, 107, 13975-13976). Inhibitors of mTOR such as rapamycin, decrease the level of phosphorylated elF4E (Hay, N. Proc. Natl. Acad. Sci. U.S.A 2010, 107, 13975-13976). The treatment with rapalogs typically leads to the clinically stable disease or partial remission rather than complete tumor regression (Gibbons, J. J.; Abraham, R. T.; Yu, K. Semin. Oncol. 2009, 36 Suppl 3, S3-S17). Combination therapy with MNK1/2 and mTOR kinases inhibitors could be a viable strategy to treat certain types of cancer (Wang, X.; Yue, P.; Chan, C. B.; Ye, K.; Ueda, T.; Watanabe-Fukunaga, R.; Fukunaga, R.; Fu, H.; Khuri, F. R.; Sun, S. Y. Mol. Cell Biol. 2007, 27, 7405-7413). WO 2010/055072 discloses MNK and mTOR combination therapy with small molecules, antibodies and siRNA for the treatment of cancer, and recent findings support that MNK and mTOR combination induces apoptosis in cutaneous T cell lymphoma cells (WO2010055072; Marzec, M.; Liu, X.; Wysocka, M.; Rook, A. H.; Odum, N.; Wasik, M. A. PLoS. One. 2011, 6, e24849).
Macrophages are major effectors of innate immunity, stimulated by a broad variety of bacterial products through specific TLRs on the cell surface to produce pro-inflammatory cytokines, such as TNF. E. coli LPS is a potent stimulus to macrophage gene expression, especially TNF, by engaging the TLR4 membrane signaling complex (Hou, L.; Sasaki, H.; Stashenko, P. Infect. Immun. 2000, 68, 4681-4687). It was shown that TLR signaling pathways require MNK expression through the use of a panel of commercial TLR agonist panel on macrophage. TNF production was increased as a response to Salmonella LPS (TLR4), ODN2006 (TLR9), HKLM (TLR2), FSL (TLR6/2) and imiquimod (TLR7) stimulation. In each case the production of TNF was inhibited by MNK kinases inhibitor CGP57380 in a dose dependant fashion and the release of multiple innate proinflammatory cytokines were affected, supporting a central role for MNK in inflammation (Rowlett, R. M.; Chrestensen, C. A.; Nyce, M.; Harp, M. G.; Pelo, J. W.; Cominelli, F.; Ernst, P. B.; Pizarro, T. T.; Sturgill, T. W.; Worthington, M. T. Am. J. Physiol Gastrointest. Liver Physiol 2008, 294, G452-G459).
MNK inhibitors can regulate the innate immune response in macrophage. A compound with anti inflammatory properties will inhibit the release of pro-inflammatory cytokines. It has been shown that CGP57380, a MNK inhibitor, inhibits the release of TNF alpha by macrophage (and not eIF4E). According to WO2005/003785 MNK kinases are promising targets for anti-inflammatory therapy.
MNK1/2 were also reported to phosphorylate a number of different proteins in addition to eIF4E. Three of these are hnRNPA1, cPLA2 and Sprouty2 (Guil, S.; Long, J. C.; Caceres, J. F. Mol. Cell Biol. 2006, 26, 5744-5758; Buxade, M.; Morrice, N.; Krebs, D. L.; Proud, C. G. J. Biol. Chem. 2008, 283, 57-65; Hefner, Y.; Borsch-Haubold, A. G.; Murakami, M.; Wilde, J. I.; Pasquet, S.; Schieltz, D.; Ghomashchi, F.; Yates, J. R., III; Armstrong, C. G.; Paterson, A.; Cohen, P.; Fukunaga, R.; Hunter, T.; Kudo, I.; Watson, S. P.; Gelb, M. H. J. Biol. Chem. 2000, 275, 37542-37551; DaSilva, J.; Xu, L.; Kim, H. J.; Miller, W. T.; Bar-Sagi, D. Mol. Cell Biol. 2006, 26, 1898-1907). Their role and function is still being investigated. Among these substrates, hnRNPA1 is overexpressed in colorectal cancer and could contribute to maintenance of telomere repeats in cancer cells with enhanced cell proliferation (Ushigome, M.; Ubagai, T.; Fukuda, H.; Tsuchiya, N.; Sugimura, T.; Takatsuka, J.; Nakagama, H. Int. J. Oncol. 2005, 26, 635-640). It is also reported that the expression levels of hnRNPA/B is deregulated in non small cell lung cancer (Boukakis, G.; Patrinou-Georgoula, M.; Lekarakou, M.; Valavanis, C.; Guialis, A. BMC. Cancer 2010, 10, 434).
MNK inhibitors have a substantial potential for the treatment of cancers including breast, prostate, hematological malignancies (e.g., CML, AML), head and neck, colon, bladder, prostatic adenocarcinoma, lung, cervical, and lymphomas (Soni, A.; Akcakanat, A.; Singh, G.; Luyimbazi, D.; Zheng, Y.; Kim, D.; Gonzalez-Angulo, A.; Meric-Bernstam, F. Mol. Cancer Ther. 2008, 7, 1782-1788; Berkel, H. J.; Turbat-Herrera, E. A.; Shi, R.; De Benedetti, A. Cancer Epidemiol. Biomarkers Prev. 2001, 10, 663-666; Wendel, H. G.; De Stanchina, E.; Fridman, J. S.; Malina, A.; Ray, S.; Kogan, S.; Cordon-Cardo, C.; Pelletier, J.; Lowe, S. W. Nature 2004, 428, 332-337; De Benedetti, A.; Graff, J. R. Oncogene 2004, 23, 3189-3199).